GOF Mutation Research Articles

Clinical Spectrum of Autoimmune Lymphoproliferative Syndrome Associated with Caspase10 Mutations (ALPS-CASP10)

Autoimmune lymphoproliferative syndrome (ALPS) is a rare disorder of the immune system due to defective Fas-mediated apoptosis which usually presents in childhood with lymphadenopathy, hypersplenism, hypergammaglobulinemia and multilineage cytopenias. Heterozygous germline mutations in FAS (TNFRSF6) gene commonly cause what is known as ALPS-FAS. Patients with genetic defect in Caspase 10 are classified as ALPS-CASP10. We have identified 16 individuals from 11 families with caspase 10 mutations. However six individuals were noted to have recognized caspase 10 polymorphisms. Here we are describing the clinical spectrum of a cohort of 10 patients from 8 families seen in our institution during the last 20 years with features suggestive of ALPS due to Caspase 10 mutations.There were 5 males and 5 females. Their median age at presentation was 7 years (Range 1-32yrs). Lymphadenopathy (n=8) was noted in most and splenomegaly in 5/10. The immunophenotypic signature of ALPS, "double negative" TCR-__+ T lymphocytes (DNTs) that lack both CD4 and CD8 markers were expanded in 6/8 patients. One patient (#132.1) had significant autoimmune kidney disease (glomerulonephritis) in addition to AIHA and ITP requiring renal transplantation. Patient #11.1 had acquired factor VIII inhibitor, hematuria, joint effusions and needed long term immunosuppression including weekly methotrexate. She is currently off all immunosuppressive medications and without any clinically significant autoimmune problems. More details of the clinical spectrum seen in this cohort of patients are presented in Table 1.During follow up we noted significant hypogammaglobulinemia requiring IVIG associated with infiltrative lung lesions and arthropathies in two siblings from family #171. Their unique evolving clinical phenotype led to diagnosis of a second genetic defect (STAT3 gain of function defect: 2141C>T; p.P714L) in them. The Caspase 10 mutation seen in this family(pI406L) was also noted following EXOME sequencing in another child with congenital neurological findings and her healthy parent as well as healthy sibling. Hence we are attributing the more recent clinical findings of hypogammaglobulinemia, diffuse lymphocytic lung disease and pulmonary fibrosis in patients 171.1.and 171.4 to the recently described STAT3 GOF mutation (Reference). Lesson learnt from this one family we wish to share is that if evolving clinical phenotype in a patient does not fit the working diagnosis of a known monogenic disorder, it is worthwhile to expand the search for other coexisting genetic defects using extended genetic testing methods currently in vogue.Reference:Milner JD, Vogel TP, Forbes L, Ma CA, Stray-Pedersen A, Niemela JE, Lyons JJ, Engelhardt KR, Zhang Y, Topcagic N, Roberson ED, Matthews H, Verbsky JW, Dasu T, Vargas-Hernandez A, Varghese N, McClain KL, Karam LB, Nahmod K, Makedonas G, Mace EM, Sorte HS, Perminow G, Rao VK, O'Connell MP, Price S, Su HC, Butrick M, McElwee J, Hughes J, Willet J, Swan D, Xu Y, Santibanez-Koref M, Slowik V, Dinwiddie DL, Ciaccio CE, Saunders CJ, Septer S, Kingsmore SF, White AJ, Cant AJ, Hambleton S, Cooper MA. Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations. Blood. 2014 Oct 30.PMID: 25359994. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chronic mucocutaneous candidiasis: characterization of a family with STAT-1 gain-of-function and development of an ex-vivo assay for Th17 deficiency of diagnostic utility.

SummaryChronic mucocutaneous candidiasis (CMC) is characterized by recurrent and persistent superficial infections, with Candida albicans affecting the mucous membranes, skin and nails. It can be acquired or caused by primary immune deficiencies, particularly those that impair interleukin (IL)−17 and IL‐22 immunity. We describe a single kindred with CMC and the identification of a STAT1 GOF mutation by whole exome sequencing (WES). We show how detailed clinical and immunological phenotyping of this family in the context of WES has enabled revision of disease status and clinical management. Together with analysis of other CMC cases within our cohort of patients, we used knowledge arising from the characterization of this family to develop a rapid ex‐vivo screening assay for the detection of T helper type 17 (Th17) deficiency better suited to the routine diagnostic setting than established in‐vitro techniques, such as intracellular cytokine staining and enzyme‐linked immunosorbent assay (ELISA) using cell culture supernatants. We demonstrate that cell surface staining of unstimulated whole blood for CCR6+CXCR3–CCR4+CD161+ T helper cells generates results that correlate with intracellular cytokine staining for IL‐17A, and is able to discriminate between patients with molecularly defined CMC and healthy controls with 100% sensitivity and specificity within the cohort tested. Furthermore, removal of CCR4 and CD161 from the antibody staining panel did not affect assay performance, suggesting that the enumeration of CCR6+CXCR3–CD4+ T cells is sufficient for screening for Th17 deficiency in patients with CMC and could be used to guide further investigation aimed at identifying the underlying molecular cause.

Abstract LB-84: Mutant p53 enhances the metastatic potential of ovarian cancer cells

Abstract Missense-mutations of TP53 resulting in accumulation of mutant proteins are very frequent in human cancer including ovarian cancer. Aggressive ovarian cancer is characterized by a peritoneal metastasis which involves the adhesion, migration, and invasion of tumor cells to the peritoneal cavity. In the present study, we investigated the effect of p53 GOF mutation (p53R248) on the metastatic potential of human ovarian cancer cells. We found that forced overexpression of p53R248 significantly increased adhesion to mesothelial cell Met5A, migration, and invasion in p53-null SKOV-3 cells. Conversely, knockdown of mutant p53 using siRNA significantly compromised p53R248-induced cell adhesion, migration, and invasion. Microarray analysis revealed that several metastasis-related genes including integrins and S1PR1(Sphingosine-1-phosphate receptor 1), and certain signaling pathway including PI3K/AKT were markedly up-regulated in p53R248 transfectant SKOV-3R248 cells. Inhibition of PI3K/Akt signaling using specific inhibitor significantly attenuated p53R248-mediated mesothelial cell adhesion, migration, and invasion. These finding show for the first time that mutant p53R248 may exert gain-of-function activity to regulate metastasis-related gene expression and the PI3K/Akt singling pathway in ovarian cancer. Citation Format: Ji-Hye Ahn, Jong-Gyu Lee, Jung-Hye Choi. Mutant p53 enhances the metastatic potential of ovarian cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-84. doi:10.1158/1538-7445.AM2014-LB-84

Abstract 3422: Strategies for molecularly enhanced chemotherapy in endometrial tumors with mutant p53 .

Abstract Serous uterine endometrial carcinomas are aggressive type II cancers with a poor outcome and for which new treatment strategies are urgently needed, in particular strategies that augment sensitivity to the established chemotherapy regimen of carboplatin and paclitaxel. The tumor suppressor gene TP53 is dysregulated in more than 50% of serous tumors. Distinct p53 mutations have different properties such that some mutations result in expression of a non-functional protein, whereas others alter binding to transcriptional targets to yield a “gain-of-function” (GOF) oncogenic protein. Our previous work revealed that endometrial cancer cells with inactivated p53 but not GOF p53 are exquisitely sensitive to treatments that abrogate the G2/M checkpoint, such as the combination of an EGFR inhibitor and paclitaxel. Since endometrial cancer cells express multiple angiogenic tyrosine kinase receptors, we hypothesized that anti-angiogenic agents also induce G2/M arrest and mitotic catastrophe when combined with paclitaxel in endometrial cancer cells with non-functional p53. This study utilizes BIBF1120, an investigational VEGFR, PDGFR, and FGFR multi-tyrosine kinase inhibitor with established anti-angiogenic activity in the vascular endothelium. Using endometrial cancer cells with either non-functional or GOF mutations in p53, we examined the impact of combining BIBF1120 and paclitaxel on expression of G2/M checkpoint controllers, cell cycle progression, and cell viability. . When combined with paclitaxel, BIBF1120 abrogated the G2/M checkpoint in p53-null endometrial cancer cells but not in cells with p53 GOF mutation. The mechanism of synergy in the p53-null cells included activation of G2/M checkpoint regulators Cdc25C, Wee1, Myt1 and Cdc2, followed by induction of mitotic cell death. Interestingly, paclitaxel-resistant cells with loss of functional p53 retained sensitivity to the BIBF1120 and paclitaxel combination treatment. In endometrial cancer cells with p53 GOF mutation, sensitivity to the paclitaxel and BIBF1120 combination was restored by the histone deacetylase inhibitor (HDAC) LBH589 or Wee-1 inhibitor MK-1775. These findings reveal that, in addition to anti-angiogenic activity, the angiokinase inhibitor BIBF1120 restores sensitivity to paclitaxel and induces mitotic cell death by targeting multiple growth factor receptors expressed in endometrial cancer cells with p53 loss of function mutation. Our data also identify novel strategies for overriding the G2/M checkpoint and achieving chemosensitization in cells with the oncogenic p53 GOF mutation. These preclinical data serve as a critical platform for the creative design of future clinical trials utilizing molecularly enhanced chemotherapy. Citation Format: Xiangbing Meng, Shujie Yang, Danlin Zhu, Xinjun Wang, Kristina W. Thiel, Kimberly K. Leslie. Strategies for molecularly enhanced chemotherapy in endometrial tumors with mutant p53 . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3422. doi:10.1158/1538-7445.AM2013-3422

Abstract 3195: Genomic amplification of IGF-1R in osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. Even though the current treatment regimen has increased event-free survival to 70%, there still exists a poor prognosis for patients with unresectable primary tumors and those with clinically evident metastases. Insulin like growth factor-1 (IGF-1) and its receptor are thought to have a role in the pathogenesis, invasion, and distant metastasis of many human solid tumors. Our previous preclinical data using an IGF-1R antibody against xenograft models of OS demonstrated responses in four out of six OS xenografts suggesting this as a potential therapeutic target. It may be hypothesized that IGF-1R is selected for genomic amplification in some OS due to its proliferative and anti-apoptotic effects. Real time RT-PCR was performed on the primary patient samples, xenografts, and 143B, a standard cell line, to see the expression of IGF-1R. Only 3 of 20 patient samples, and 1 of 4 xenografts have shown higher level of expression of IGF-1R in reference to GAPDH. Genomic DNA was isolated from the same samples, and quantitative PCR was performed to detect gene copy numbers. 11 of 19 (58%)samples were shown to have more than 2 copies of IGF-1R (2 samples have >10 copies, 1 has 7, 2 have 5, 6 have 33), whereas 37% have 2 copies. Two OS xenografts, which respond to IGF-1R antibody in preclinical testing, showed increases in the copy number of IGF-1R. Fluorescence in situ hybridization (FISH) was used to detect the amplification of IGF-1R gene in both patient derived and xenograft OS cell lines. FISH was performed on metaphase slides of fixed cultured cells. Three BAC probes spanning the IGF-1R gene locus in 15q26.3 were produced by isolating BAC DNA, and labeling with AlexaFluor 488 by nick translation. Metaphase prep slides were then hybridized with IGF-1R probes and Texas Red-labeled chromosome 15 centromere probes, counter stained with DAPI, and viewed under fluorescence microscope. Copy number was counted in cells showing a full metaphase spread. Based on initial FISH results, it seems that in the majority of OS the IGF-1R gene is not amplified. This does not imply that IGF1R overamplification does not play a role in IGF-IR overexpression in OS: given the heterogeneity of these tumors and the limited sample size of the experiments thus far IGF-1R amplification may be a rare phenomenon in OS. As responses to IGF-1R are not observed in the majority, it remains possible that amplification correlates with therapeutic response to IGF-1R antibody. FISH on more patient cell lines and xenografts are underway, and even if no amplification is demonstrated, increased IGF-IR activity could still be explained by GOF mutation, or other mechanisms. Additional experiments including FISH in a larger pool of samples, comparing amplification results to clinical data and responsiveness of the cell lines to anti-IGF-IR therapy, and looking for other abnormalities in the IGF-I system, among others are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3195.

Elevated expression of p53 gain-of-function mutation R175H in endometrial cancer cells can increase the invasive phenotypes by activation of the EGFR/PI3K/AKT pathway

Backgroundp53 is the most commonly mutated tumor suppressor gene in human cancers. In addition to the loss of tumor suppression function and exertion of dominant-negative effects over the remaining wild-type protein, several p53 mutants can gain novel oncogenic functions (gain-of-function, GOF) that actively regulate cancer development and progression. In human endometrial cancer, p53 mutation is more often associated with aggressive nonendometrioid cancer. However, it was unknown if p53 mutants contributed to endometrial cancer progression through the GOF properties.MethodsTo clarify the relationship between expression of p53 GOF mutation (p53-R175H) and invasive potential of human endometrial cancer KLE cells, we tested the consequences of up-regulation and down-regulation of p53-R175H in KLE cells by inducing p53-R175H expression vector or suppressing the p53 gene with short hairpin RNA.ResultsWe found that forced over-expression of p53-R175H significantly promoted cell migration and invasion, and induced activation of the epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/AKT pathway. Conversely, suppression of p53-R175H with short hairpin RNA significantly inhibited cell migration and invasion, and resulted in attenuation of EGFR/PI3K/AKT pathway.ConclusionThese findings show for the first time that elevated expression of p53-R175H mutant may exert gain-of-function activity to activate the EGFR/PI3K/AKT pathway and thus may contribute to the invasive phenotype in endometrial cancer.